Most locally advanced solid tumors contain regions of reduced oxygen availability (Vaupel and Mayer, Cancer Metastasis Rev. 26: 225-339; Semenza G L, Drug Discovery Today 12: 853-859). Intratumoral hypoxia results when cells are located too far from a functional blood vessel for diffusion of adequate amounts of oxygen (O2), as a result of rapid cancer cell proliferation and the formation of blood vessels that are structurally and functionally abnormal. In the most extreme case, O2 concentrations are below those required for survival, resulting in cell death and establishing a selection for cancer cells in which apoptotic pathways are inactivated, anti-apoptotic pathways are activated, or invasion/metastasis pathways that promote escape from the hypoxic microenvironment are activated. This hypoxic adaptation may arise by alterations in gene expression or by mutations in the genome or both and is associated with decreased patient survival (Ibid.).
In addition to this intratumoral role for hypoxia, systemic, local, and intracellular homeostatic responses elicited by hypoxia include erythropoiesis by individuals who are anemic or at high altitude (Jelkmann (1992) Physiol. Rev. 72:449-489), neovascularization in ischemic myocardium (White et al. (1992) Circ. Res. 71:1490-1500), and glycolysis in cells cultured at reduced oxygen tension (Wolfle et al. (1983) Eur. J. Biochem. 135:405-412). These adaptive responses either increase oxygen delivery or activate alternate metabolic pathways that do not require oxygen. Hypoxia-inducible gene products that participate in these responses include erythropoietin (EPO) (reviewed in Semenza (1994) Hematol. Oncol. Clinics N. Amer. 8:863-884), vascular endothelial growth factor (Shweiki et al. (1992) Nature 359:843-845; Banai et al. (1994) Cardiovasc. Res. 28:1176-1179; Goldberg & Schneider (1994) J. Biol. Chern. 269:4355-4359), and glycolytic enzymes (Firth et al. (1994) Proc. Natl. Acad. Sci. USA 91:6496-6500; Semenza et al. (1994) J. Biol. Chern. 269: 23757-23763; Semenza U.S. Pat. No. 5,882,914).
Investigation of the molecular regulation of hypoxia and the EPO gene, which encodes a growth factor that regulates erythropoiesis and thus blood oxygen carrying capacity (Jelkmann (1992) supra; Semenza (1994) supra), resulted in identification of cis-acting DNA sequences in the EPO 3′-flanking region required for transcriptional activation in response to hypoxia. HIF-1 (hypoxia inducible factor 1) was identified as a trans-acting factor that binds to this enhancer. Previously known inducers of EPO expression (1% oxygen, cobalt chloride (CoCl2) and desferrioxamine (DFX or, alternatively, DFO herein)) also induced HIF-1 DNA binding activity with similar kinetics; inhibitors of EPO expression (actinomycin D, cycloheximide, and 2-aminopurine) blocked induction of HIF-1 activity; and mutations in the EPO 3′-flanking region that eliminated HIF-1 binding also eliminated enhancer function (Semenza (1994) supra).
HIF-1 is a dimer composed of HIF-1α and HIF-1β subunits. HIF-1α is a basic helix-loop-helix (bHLH) transcription factor encoded by the HIF1A gene (Semenza et al., Genomics 34: 437-9; Hogenesch et al., J. Biol. Chem. 272: 8581-93). While the HIF-1β subunit is constitutively expressed, the HIF-1α subunit is the limiting member of the heterodimer and therefore regulates HIF-1 levels. Under conditions of normal oxygen, HIF-1α is ubiquinated and rapidly degraded. However, under hypoxic conditions the rate of ubiquitination dramatically decreases and HIF-1α is stabilized, resulting in upregulation of HIF-1 dimer. This is an important point and provides a rationale for targeting HIF-1α instead of HIF-1β for modulating HIF-1 activity (Akinc et al., U.S. Pat. No. 7,737,265).
Notably, HIF-1α overexpression has been associated with increased patient mortality in a variety of cancers (Semenza G L, Drug Discovery Today 12: 853-859), and a role for HIF-1α has also been described, e.g., for both “wet” and “dry” forms of age-related macular degeneration (AMD; see Akinc et al., supra).
Double-stranded RNA (dsRNA) agents possessing strand lengths of 25 to 35 nucleotides have been described as effective inhibitors of target gene expression in mammalian cells (Rossi et al., U.S. Patent Application Nos. 2005/0244858 and US 2005/0277610). dsRNA agents of such length are believed to be processed by the Dicer enzyme of the RNA interference (RNAi) pathway, leading such agents to be termed “Dicer substrate siRNA” (“DsiRNA”) agents. Additional modified structures of DsiRNA agents were previously described (Rossi et al., U.S. Patent Application No. 2007/0265220).